1. Field of the Invention
This invention relates to the field electrophoresis using multiple recovery cells for cast gels. 2. Prior Art
The prior art is replete with descriptions of apparatus and empirical results of the practice of electrophoresis. Electrophoresis can generally be defined as the movement in an electric field of charged molecules in any substance, typified by inorganic ions, proteins, enzymes, and blood serum. The basic operating principle is that any charged particle suspended between the poles of an electrical field tends to travel toward the pole that bears the charge opposite to its own. The rate of travel is conditioned by a variety of factors including the characteristics of the individual particle, the properties of the electrical field and operating parameters such as temperature and the nature of the suspending medium or buffer.
These fundamental principles of electrophoresis have been known since the 1920's [See: "Advances In Protein Chemistry" edited by M. L. Arsen and John T, Edsall, Vol. IV, pp. 272- 277 (1948)].
While the subject of much academic interest, the practical application of these electrophorectic principles presented many practical difficulties such as leakage, heat generation and the inordinate length of time needed for yielding meaningful results. The mobilities of molecules under study can be established only with experimental data under reproducible circumstances and, accordingly, the control of environment and operating conditions have been a major hurdle to overcome in the prior art. An early solution is typified in U.S. Pat. No. 2,758,966 wherein in a dispersion a horizontal electrical field is created which causes a horizontal migration of colloid particles in the dispersion. This horizontal migration proceeds until vertical barriers of semipermeable membranes are reached where the migration is stopped and the particles accumulate. The differences in density in the dispersion near the membrane as a result of the accumulation of the charged particles creates a convection current transporting distinct components at different rates thereby accomplishing the desired separation. This type of device typifies so-called free-solution electrophoresis and the prior art contains a variety of such devices such as in U.S. Pat. Nos. 3,047,489 and 3,129,158.
Another technique of importance in the prior art is that of providing a bed or support onto which the electrophorectic process can take place both for analysis and separation. An early technique was to use filter paper, however, there have been problems of diffusion and absorption of the molecules to the paper. This generally results in a loss of definition in separation as well as losses of 10- 15 percent in accumulated content (See: U.S. Pat. No. 2,555,487). U.S. Pat. No. Re. 24,752 sought to minimize these losses by using a fluid film as the medium. A gelling agent is employed in the buffer such that although the buffer is a liquid, at small angles of tilt, if applied to a glass plate, there would be no movement of fluid. In practice, this film is placed on a glass plate and the particular composition to be analyzed is added by placing a small piece of filter paper which has been soaked in the material onto one end of the plate and the electric field is applied. While techniques such as this are suitable for analytical electrophoresis they are totally not adaptable for the collection of the isolated component fraction.
The prior art has also attempted to accomplish separation by the use of supporting gels as typified by U.S. Pat. No. 3,450,624. A large amount of gel is used as the support medium, cast vertically, to which the sample is added and electrophoresis takes place horizontally across the gel with eludes collected at the base. Devices such as in this patent rely on long running times to accomplish separation with the attendant problems of heating. It is evident that maintaining uniform temperatures during long runs becomes a governing criteria which has heretofore made gel support mediums generally unsatisfactory for elution or collection electrophoresis. Attempts to minimize these difficulties have resulted in the use of cut pieces of gel on a screen with elution taking place with a bag composed of dialysis tubing [Haschke and Campbell, J. Bac. 105,249 (1971)] for sample elution. Similarly, U.S. Pat. No. 3,255,100 describes an improved apparatus for the collection of the isolated component fraction in its pure state without the presence of other substances such as the supporting medium.
According to the teachings of the Raymond patent, after one electrophoresis run in which the components of a complex substance are separated on the supporting gel, this gel is then placed in an electrophoresis cell with the gel oriented vis-a-vis the electrodes such that migration occurs under the influence of the electric field with the fractions maintained separately during movement by a separating block. When the dialysis membrane is reached a series of vertical channels allow the accumulation by gravity in a convection current of the separated components into suitable collection receptacles.
Devices such as Raymond allow for the collection of individual fractions from a single gel, however, the elution of a number of separate fractions has heretofore not been feasible.